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Understanding and Exploiting MYCN Addiction in High-Risk Neuroblastoma Using the Example of CDK13

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Understanding and Exploiting MYCN Addiction in High-Risk Neuroblastoma Using the Example of CDK13 Understanding and Exploiting MYCN Addiction in High-Risk Neuroblastoma Using the Example of CDK13 Dissertation Mona Soledad Friedrich, M. Sc. Biowissenschaften Dissertation submitted to the Combined Faculty of Natural Sciences and Mathematics of the Ruperto Carola University Heidelberg, Germany for the degree of Doctor of Natural Sciences Presented by Mona Soledad Friedrich, M.Sc. Born in: Karlsruhe Oral Examination: 03.06.2019 Understanding and Exploiting MYCN Addiction in High-Risk Neuroblastoma Using the Example of CDK13 Referees: Prof. Dr. Thomas Höfer PD Dr. Frank Westermann Sperrvermerk für die Dissertation „Understanding and Exploiting MYCN Addiction in High-Risk Neuroblastoma Using the Example of CDK13” von Mona Soledad Friedrich, M. Sc. Diese Arbeit enthält vertrauliche Daten und Informationen des Deutschen Krebsforschungszentrums und muss deshalb bis zum 11.10.2020 gegen Veröffentlichung gesperrt werden. Während dieses Zeitraumes sind Veröffentlichungen oder Vervielfältigungen – auch nur auszugsweise – ohne ausdrückliche schriftliche Genehmigung des Deutschen Krebsforschungszentrums nicht gestattet. Alle Rechte zum Erwerb und zur Anmeldung gewerblicher Schutzrechte sind dem Deutschen Krebsforschungszentrum vorbehalten. Declaration The work presented in this thesis was carried out from March 2014 until March 2019 in the group of Neuroblastoma Genomics at the German Cancer Research Center (DKFZ) in Heidelberg, Germany. It was supervised by PD Dr. Frank Westermann. I declare that this thesis, and the research to which it refers, are the product of my own work that has not yet been submitted for a degree or a diploma at any university. To the best of my knowledge this thesis contains no material previously published or written by another person except where due acknowledgement is made in the thesis itself. _____________________ Mona Friedrich Heidelberg, April 10, 2019 SUMMARY Neuroblastoma (NB) is an extracranial childhood cancer and accounts for 15 % of all childhood cancer-related deaths. It is a remarkably heterogeneous disease and cases range from spontaneous regression to aggressive high risk disease. Amplification of the oncogene MYCN occurs in 20-30 % of cases and is an indicator of poor prognosis. As a transcription factor, MYCN drives several cellular programs favoring malignant behavior, for example proliferation, angiogenesis and metastasis. In order to gain a better understanding of direct and secondary transcriptional targets of MYCN, gene expression was analyzed in a time course experiment using cell cycle-synchronized cells with regulatable MYCN level. This approach revealed the continued upregulation of a number of cell cycle driver genes as well as genes involved in protein and DNA biosynthesis in MYCN high cells. Differences in the expression of other cell cycle and DNA replication genes occurred only in specific phases of the cell cycle. On the other hand, genes involved in alternative splicing and cell adhesion were constantly downregulated. The expression of snoRNAs strongly increased in the MYCN low condition towards the end of the observation period. An analysis of miRNA expression revealed that many differentially regulated miRNAs targeted genes involved in ribosome biogenesis, cancer processes and signaling pathways. Taken together, this data set indicates that MYCN directly regulates the expression of genes and miRNAs which contribute to accelerated proliferation, metabolism and metastatic growth in NB cells. As a consequence of the induced phenotype, a larger number of secondary targets are deregulated. As MYCN heavily contributes to tumor malignancy, MYCN-amplified NB cells become addicted to high amounts of the protein. However, MYCN itself is notoriously difficult to target, therefore two siRNA screens were performed to detect synthetic lethal relationships with high MYCN levels. The second part of this thesis deals with the transcriptional kinase CDK13, which was identified as a potential candidate for novel targeted therapies. CDK13 and its highly homologous family member CDK12 were knocked down by several technical approaches. In a cellular MYCN overexpression model, CDK13 repression induced strong cell death only in MYCN high cells. CDK12 repression also elicited a small amount of cell death comparing MYCN high with low cells. Inducible CDK13 knockdown in a MYCN-amplified cell line caused modest reductions in cellular viability and colony formation capacity. CDK13, but not CDK12, knockout induced by the clustered regularly interspaced short palindromic repeats (CRISPR) technique reduced viability and caused a small increase in cells arrested in G1. However, analysis of CDK13 mRNA level revealed significant residual expression, suggesting that the majority of the polyclonal culture might be heterozygous for the induced mutation. A novel small I SUMMARY compound inhibitor against CDK12 and CDK13 (BAY-587) was tested in a panel of eight NB cell lines and the effects were compared to that of a commercially available inhibitor, THZ531. BAY-587 was active at lower concentrations than THZ531. Both compounds strongly reduced viability, colony formation capacity and disrupted cell cycle distributions. BAY-587 treatment reduced the level of CDK12 protein and further induced apoptosis. Gene expression analysis revealed that CDK12/13 inhibition by BAY-587 caused downregulation of genes involved in alternative splicing and DNA damage repair, while transcription regulation genes were upregulated. In summary, CDK13 emerged as a promising new therapeutic candidate for the treatment of high risk NB patients. II ZUSAMMENFASSUNG Das Neuroblastom ist ein extrakranialer Tumor, der im Kindesalter auftritt und für 15 % der Todesfälle im Zusammenhang mit Krebserkrankungen bei Kindern verantwortlich ist. Der Verlauf dieser Erkrankung ist sehr heterogen, es gibt Fälle, bei denen sich der Tumor spontan zurückbildet, aber auch sehr aggressive Hochrisikofälle. In 20 – 30 % der Fälle liegt eine Amplifizierung des Onkogens MYCN vor, was mit einer deutlich schlechteren Prognose verbunden ist. MYCN ist ein Transkriptionsfaktor und reguliert verschiedene zelluläre Programme, die bösartiges Wachstum begünstigen, wie zum Beispiel Proliferation, Angiogenese und Metastasierung. Um mehr über direkte und indirekte transkriptionelle Ziele von MYCN zu erfahren, wurde die Genexpression synchronisierter Zellen mit regulierbarem MYCN Level über einen längeren Zeitraum gemessen. Dabei wurden Gene identifiziert, die über die gesamte Beobachtungsdauer hinweg stärker in Zellen mit viel MYCN Protein exprimiert wurden. Darunter fanden sich mehrere Zellzyklusgene sowie vermehrt Gene der Protein- und DNA-Biosynthese. Bei einigen anderen Zellzyklusgenen und DNA Replikationsgenen unterschied sich die Expression in Zellen mit viel oder wenig MYCN Protein nur in spezifischen Zellzyklusphasen. Im Gegensatz dazu wurden Gene des alternativen Spleißens und der Zelladhäsion in Zellen mit viel MYCN konstant niedriger exprimiert. In Zellen mit wenig MYCN gab es zudem einen starken Anstieg in der Expression von snoRNAs gegen Ende des Beobachtungszeitraumes. Eine Analyse der miRNA Expression ergab, dass MYCN vorwiegend miRNAs beeinflusst, welche Gene der Ribosom Biogenese, Tumorbiologie und Signalwege regulieren. Zusammenfassend zeigt der vorliegende Datensatz, dass MYCN durch die direkte Regulierung von Gen- und miRNA-Expression zu beschleunigter Proliferation, Metabolismus und metastasierendem Wachstum beiträgt. Der daraus resultierende Phänotyp bedingt die Deregulierung weiterer, sekundärer Zielgene. Da MYCN einen großen Anteil an der Bösartigkeit des Tumors trägt, werden MYCN- amplifizierte Neuroblastomzellen abhängig von großen Mengen des Proteins in der Zelle. Bislang ist es problematisch, MYCN selbst durch Wirkstoffe anzugreifen. Aus diesem Grund wurden zwei siRNA Screens durchgeführt, die zum Ziel hatten, synthetisch-letale Interaktionen mit erhöhtem MYCN Protein zu ermitteln. Der zweite Teil dieser Arbeit beschäftigt sich mit der Charakterisierung eines Kandidaten aus diesen Screens, der transkriptionellen Kinase CDK13. Knockdowns von CDK13 sowie einer verwandten Kinase aus derselben Proteinfamilie, CDK12, wurden mittels verschiedener Techniken erzielt. In einem Zellmodell zur Überexpression von MYCN führte der Knockdown von CDK13 zu starkem sowie der Knockdown von CDK12 zu leichtem Zelltod. CDK13 Knockdown in einer MYCN-amplifizierten Zelllinie führte zu einer leichten Verminderung III ZUSAMMENFASSUNG von Viabilität und der Fähigkeit, aus Einzelzellen Kolonien zu bilden. Darüber hinaus wurde mittels CRISPR (clustered regularly interspaced short palindromic repeats) ein Knockout von CDK13 erzielt, was zu einer Reduktion der Viabilität sowie einem leichten G1-Arrest führte, nicht jedoch bei einem Knockout von CDK12. Allerdings war die CDK13 mRNA Expression um weniger als 50 % reduziert, was auf das Vorliegen einer polyklonalen Zellkultur mit vermutlich vorwiegend heterozygoten Mutationen zurückzuführen war. Des Weiteren wurde ein neuartiger Inhibitor von CDK12 und CDK13 (BAY-587) in acht verschiedenen Neuroblastom Zelllinien getestet und mit dem kommerziell erhältlichen Inhibitor THZ531 verglichen. Alle Zelllinien reagierten sensitiver auf BAY-587 als auf THZ531 und wiesen niedrigere IC50 Werte auf. Beide Inhibitoren verminderten die Zellviabilität und die Fähigkeit, Kolonien zu bilden und riefen Veränderungen der Zellzyklusprofile hervor. Die Behandlung mit BAY-587 verminderte den CDK12 Proteingehalt und induzierte außerdem Apoptose.
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